The Role of DNA Methylation in Human Disease – Technology Networks

DNA methylation is one of the earliest epigenetic modifications to be discovered in human beings. It involves the transfer of methyl (CH3) groups to the C5 position of cytosine bases that comprise deoxyribonucleic acid (DNA) to produce 5-methylcytosine (5mC) the reaction is catalyzed by a family of enzymes called DNA methyltransferases (DNMTs). Typically, the altered cytosine bases reside immediately adjacent to guanine bases. This leads to two 5mC bases sitting diagonally to each other on complementary DNA strands.DNMTs have several distinct roles, for instance, they may function asde novoDNMTs, which involves establishing the initial pattern of methyl groups on a DNA molecule. While other DNMTs adopt maintenance roles, copying the methylation pattern from an existing DNA strand to its new partner after replication has occurred.

Several studies in the 1980s revealed that DNA methylation played a major part in both gene regulation and cell differentiation. Since then, further research has confirmed the role of abnormal methylation in the development and progression of various diseases. According to Manel Esteller, director of the Josep Carreras Leukaemia Research Institute and professor of genetics at the University of Barcelona, DNA methylation is one of the main controllers for specific-tissue expression allowing the correct expression of a gene in the right organ or cell type. He further added, DNA methylation acts as a buffer to stabilize our genome and silence repetitive chromosomic regions. Many diseases show an alteration of DNA methylation that disrupts cellular activity. Estellers research mainly focuses on alterations in DNA methylation, histone modifications and chromatin in human cancer. At present, he is working on establishing epigenome and epitranscriptome maps for normal and transformed cells.

In mammals, methylation is mostly sparse but is globally distributed in specific CpG or CG (cytosineguanine) sequences. In certain regions of the genome, CpG is abundantly found (e.g., CpG islands). In healthy cells, CpG islands associated with gene promotors are typically free from methylation, whereas islands found within gene bodies tend to become methylated during development. Researchers have pointed out that methylation of CpG islands at promotor regions can cause inappropriate downregulation of specific genes (e.g., silencing of tumor suppressor genes in cancer cells).

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DNA methylation plays an important role in many biological processes, for example, genomic imprinting, stem cell differentiation and chromosomal stability,and is considered an essential modification that regulates cell growth and proliferation. DNA methylation patterns are mutable and inheritable and in the case of abnormal DNA methylation in the parental allele, various serious diseases, such as cancer, aging disorders, metabolic ailments, psychological disorders and genetic diseases, may occur.

Systemic lupus erythematosus (SLE) is an autoimmune disease in which the bodys immune system incorrectly attacks its own healthy tissue. A genome-wide assessment of DNA methylation demonstrated differential DNA methylation in the genes of SLE patients, associated with autoantibody production. Abnormal DNA methylation was observed in the promoter region of the IL-6 gene.

In cancer, we observe a general global DNA hypomethylation of the genome and more focal DNA hypermethylation that affects CpG-rich sequences (so-called CpG islands) often found at the promoter, explained Professor Gerd Pfeifer, from the Center for Epigenetics, Van Andel Institute. Pfeifers laboratory investigates the underlying mechanisms of cancer and other diseases, specifically focusing on DNA mutations, DNA methylation and the role of 5mC oxidation. According to Pfeifer, most of the DNA hypermethylation events in cancer are inconsequential because the genes are already silent. However, some methylation events can be considered tumor drivers, when, for example, they silence genes encoding anti-proliferative factors, DNA repair genes, or genes essential for normal cell differentiation.

Paula Esteller-Cucala is a doctoral researcher in the Comparative Genomics Group at the Institut de Biologia Evolutiva (IBE), her work focuses on epigenetics and transcriptomics of non-human primates. She said,Methylation patterns are very heterogeneous. They might differ from one cancer type to another and also from one cell type to another cell type. Understanding the role of these modifications and their effect in different cancer types is essential to target potential treatments and therapies. Identifying cancer-specific DNA methylation markers (regions of the genome that are specifically methylated or unmethylated in one or more cancer types or subtypes), can be used to detect and monitor cancer with a view to developing therapeutic strategies.

Gliomas are a common type of brain cancer,which originate in the glial cells that support neurons in the brain. Recently, researchers have used a single-cell multiomics approach to identify methylation marks within individual tumor cells obtained from patients with glioma. They were able to confirm distinct patterns of DNA methylation responsible for shifting the cells from one state to another (e.g., stem-cell-like states to mature states) and developed a map of cell states from the sampled tumors. The insights gained from the study could help to develop better ways to detect, stage, monitor and treat the disease.

A lowered methylation level of catechol-O-methyl transferase in peripheral blood was observed in patients with schizophrenia.

An epigenome-wide association study compared the methylation patterns of tissues from three different mammalian species to determine if Huntingtons disease is accompanied by altered DNA methylation. The researchers found that the disease was associated with profound changes to the level of DNA methylation.

A systemic review of DNA methylation in Alzheimers disease found that the APP gene encoding a protein called amyloid precursor protein which has been associated with the formation of amyloid plaques is consistently hypermethylated in brain and peripheral blood.

Looking beyond traditional methods, recent advances in sequencing and array technologies have enabled researchers to conduct detailed DNA methylation profiling, providing a comprehensive picture of its role in disease. In Esteller-Cucalas opinion, the latest methodology used to study DNA methylation is by means of long-read sequencing these technologies allow much longer sequences to be read (> 10000 bp).

Esteller, also provided his thoughts, The technology most widely used to study, in a cost-effective manner, human DNA methylation is based on DNA methylation microarrays that interrogate 850K CpG sites of our genome.

Some techniques usedto determine DNA methylation are discussed in more detail below.

An advanced sequencing-based technique known as methylation-specific PCR (MS-PCR) has been developed which avoids the complex sequencing process.

Some examples of methylation-sensitive restriction enzymes (MREs) include HpaII, BstUI, NotI and SmaI. These enzymes only cut the nonmethylated target regions and keep the methylated DNA intact. These MRE cuttings are subsequently sequenced to predict the DNA methylation levels at the genomic level. Recently, scientists have developed an advanced enzymatic digestion technique, called methylation-sensitive restriction endonuclease-PCR/southern (MS-RE-PCR).

Pfeifer pointed out some additional considerations, "One challenge is to achieve coverage of the whole mammalian genome, which has over 25 million CpG sequences that can be methylated. To perform a quantitative analysis of the methylation state of each one of these CpGs, deep sequencing coverage is required, which is still expensive. There are more affordable methods available that can be used to analyze subsets of CpGs, but these methods may miss some critical methylation changes.

For comprehensive DNA methylation studies, a large amount of DNA may be required and therefore, analysis becomes challenging when the tissue samples are scarce. Sometimes it is difficult to distinguish 5mC from 5hmC, said Esteller.

Esteller explained that from knowledge of the DNA methylation landscape of tumoral cells, three translation uses have emerged in the oncology field: The discovery of new biomarkers of the disease that can even be detected in biological fluids and allow its pathological classification; the use of DNA hypermethylation events in certain genes as predictors of response to therapies, helping cancer precision medicine; and the use of DNA methylation as a target for epigenetic drugs such as inhibitors of DNA methylation that are being used in the treatment of hematological malignancies.

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The Role of DNA Methylation in Human Disease - Technology Networks